Joint use of SAXS and NMR Annalisa Pastore Kings College London - - PowerPoint PPT Presentation

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Joint use of SAXS and NMR Annalisa Pastore Kings College London - - PowerPoint PPT Presentation

Dec 21, 2023 41 likes •536 views

Joint use of SAXS and NMR Annalisa Pastore Kings College London Scuola Normale Superiore The three brothers of Structural Biology Lele Luzzati The limits of X-ray Necessity of crystals Little information on dynamics The limits of

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Joint use of SAXS and NMR

Annalisa Pastore King’s College London Scuola Normale Superiore

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The three brothers

  • f Structural Biology

Lele Luzzati

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The limits of X-ray…

Necessity of crystals Little information on dynamics

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The limits of cryo-EM

The protein size It is expensive The samples get radiation damnaged

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A bit of NMR history

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Nuclear Magnetic Resonance

1 Gauss 23.5 Tesla Circa 500.000 times >>

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The magnetic field removes the degeneracy of the nuclear spin levels

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NMR as a radio…

Tune the frequency to observe a certain… element….

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The 1H spectrum of a protein

The position of each peak on the spectrum is called chemical shift

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A 13C spectrum

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You may gain resolution increasing the dimensions…

Like a 2D Gel

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…or using more than one nucleus

Excite one nucleus, transfer the magnetization to another, and than back to the first

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2D maps of 1H-15N correlations

As many resonances as many HN in the protein (i.e. the Nres – Npro)

The fingerprint

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Chemical shift perturbation as a tool to map interactions…

Sensitivity to the chemical environment

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Distance restraints

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The advantanges:

  • Structure determination in solution
  • Intermolecular interactions
  • Dynamics
  • Weak interactions
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Disadvantages: The linewidth is proportional to the tumbling time

We are limited in the molecular size we can afford…

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A way to solve the structure of giant proteins

Improta et al. Structure 1997 Von Castelmur et al., PNAS 2007

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A way to validate structures

Only one structure fits the data: ours!!!

Nicastro et al., J. Biomol. NMR 2006

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A way to solve the structure of molecular complexes

Prischi et al. NSMB 2010

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The Nobel prize Rita Levi Montalcini

(1901 – 2012)

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Nerve growth factor

Nerve Growth Factor (NGF) plays an important role in cognitive function, depression, inflammation, autoimmunity, histamine intolerance, western disease, pain, cancer and more. It belongs to the neurotrophin group.

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R100W mutants are painless… Painless = fearless

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The structure of NGF was solved in 1991 by Blundell and cow.

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But NGF is expressed as a precursor

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Evidence that proNGF has an independent life

ProNGF is the more abundant form in CNS tissues, mature NGF is barely detectable Cleaved pro-domain exists in vivo with uncleaved proNGF and mature NGF In HEK TrkA stable cells, proNGF binds to TrkA at a site distinct from that of NGF

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What is the structure of proNGF?

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X-ray crystallography?

No crystals!!!

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NMR of NGF?

A

NGF

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NMR of proNGF?

B

proNGF

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Comparison NGF vs proNGF

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SAXS (Small-angle X-ray Scattering)

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Validating the structures by SAXS data

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SAXS and NMR validation

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A collapsed intrinsically unfolded region

SAXS measurements revealed the proNGF is dimeric and anisometric, with the propeptide domain being intrinsically unstructured A globular ‘‘crab-like’’ and elongated shapes equally fit the scattering data

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A crab-like structure?

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proNGF mostly behaves as a collapsed structure

IscS (90kDa) proNGF (50kDa) DHFR (20kDa) NGF (13kDa) NusA (12kDa)

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A different approach

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We cut the pro-peptide (NGFpd)

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NGFpd is unfolded with helical tendency

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NGFpd is responsible for cone collapse

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Mapping the effects of NGF

  • n NGFpd and viceversa

The individual two halves interact even though weakly

Titration of unlabelled NGF into 15N NGFpd Titration of unlabelled NGFpd into 15N NGF

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Paramagnetic relaxation enhancement

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Mapping the surface of interaction

MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)

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Restrained metadynamics

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Using SAXS to restrict the MD results

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Conclusions

ProNGF has distinct properties from NGF The pro-domain has function of its own The pro-domain is unstructured but Collapses on NGF to produce a flexible semi compact structure

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Acknowledgements

Robert Yan Filippo Prischi, Salvatore Adinolfi, Rita Puglisi Antonino Cattaneo, Doriano Lamba, Francesca Paoletti Peter Konarev and Dmitri Svergun (EMBL)

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NMR SAXS

A pact of friendship...